Protein biochips are gaining in significance on an industrial scale in analytics and diagnostics and also in pharmaceutical development. Protein biochips have become established as screening tools.
Here, the rapid and highly parallel detection of a large number of specific binding analysis molecules is made possible in a single experiment. To produce protein biochips, it is necessary to have the required proteins available. In particular, protein expression libraries have become established for this purpose. The high-throughput cloning of defined open reading frames is one possibility (Heyman, P. A., Cornthwaite, P., Foncerrada, L., Gilmore, P. R., Gontang, E., Hartman, K. P., Hernandez, C. L., Hood, R., Hull, H. M., Lee, W. Y., Marcii, R., Marsh, E. J., Mudd, K. M., Patina, M. J., Purcell, T. P., Rowland, P. P., Sindici, M. L. and Hoeffler, P. P. (1999) Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. Genome Res, 9, 383-392; Kersten, B., Feilner, T., Kramer, A., Wehrmeyer, S., Possling, A., Witt, I., Zanor, N. I., Stracke, R., Lueking, A., Kreutzberger, J., Lehrach, H. and Cahill, D. J. (2003) Generation of Arabidopsis protein chip for antibody and serum screening. Plant Molecular Biology, 52, 999-1010; Reboul, J., Vaglio, P., Rual, J. F., Lamesch, P., Martinez, M., Armstrong, C. M., Li, S., Jacotot, L., Bertin, N., Janky, R., Moore, T., Hudson, J. R., Jr., Hartley, J. L. Brasch, M. A., Vandenhaute, J., Boulton, S., Endres C. A. Jenna, S., Chevet, E., Papasotiropoulos, V., Tolias, P. P., Ptacek, J., Snyder, M., Huang, R., Chance, M. R., Lee, H., Doucette-Stamm, L., Hill, D. E. and Vidal, M. (2003) C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nat Genet, 34, 35-41.; Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S. and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol, 328, 575-592). However, such an approach is closely linked to the progress of the genome sequencing projects and the annotation of these gene sequences. In addition, the determination of the expressed sequence is not always clear clue to differential splicing processes. This problem can be avoided by the use of cDNA expression libraries (Büssow, K., Cahill, D., Nietfeld, H. Bancroft, D. Scherzinger, E., Lehrach, H. and Walter, G. (1998) A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. Nucleic Acids Research, 26, 5007-5008; Büssow, K., Nordhoff, E., Lübbert, C., Lehrach, H. and Walter, G. (2000) A human cDNA library for high-throughput protein expression screening. Genomics, 65, 1-8; Holz, C., Lueking, A., Bovekamp, L., Gutjahr, C., Bolotina, N., Lehrach, H. and Cahill, D. J. (2001) A human cDNA expression library in yeast enriched for open reading frames. Genome Res, 11, 1730-1735; Lueking, A., Holz, C., Gotthold, C., Lehrach, H. and Cahill, D. (2000) A system for dual protein expression in Pichia pastoris and Escherichia coli, Protein Expr. Purif., 20, 372-378). Here, the cDNA of a specific tissue is cloned into a bacterial or eukaryotic expression vector, such as yeast. The vectors used for the expression are generally characterised in that they carry inducible promoters, with which the moment of protein expression can be controlled. In addition, expression vectors have sequences for what are known as affinity epitopes or affinity proteins, which on the one hand allow the specific detection of the recombinant fusion proteins by means of an antibody directed against the affinity epitope, and on the other hand enable specific purification via affinity chromatography (IMAC).
For example, the gene products of a cDNA expression library from human foetal brain tissue were arranged in the bacterial expression system. Escherichia coli in high-density format on a membrane and were able to be screened successfully with different antibodies. It was possible to demonstrate that the proportion of full-length proteins is at least 66%. The recombinant proteins from expression libraries could also be expressed and purified in high throughput. (Braun P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E. and LaBaer, J. (2002) Proteome-scale purification of human proteins from bacteria, Proc Natl Acad Sci USA, 99, 2654-2659; BCssow (2000) supra; tucking, A., Horn, M., Eickhoff, H., Büssow, K., Lehrach, H. and Walter, G. (1999) Protein microarrays for gene expression and antibody screening. Analytical Biochemistry, 270, 103-111). WO 99/57311 and WO 99/57312 relate in particular to such protein biochips based on cDNA expression libraries.
Furthermore, besides antigen-presenting protein biochips, antibody-presenting arrangements are also described (Pal et al (2002) Antibody arrays: An embryonic but rapidly growing technology, DDT, 7, 143-149; Kusnezow et al, (2003), Antibody microarrays: An evaluation of production parameters, Proteomics 254-264).
There is a high demand however to improve the diagnosis of high-affinity binders, in particular antibodies, autoantibodies and marker sequences.
Protein biochips of the type described by the applicant are already described and allow the diagnosis of illnesses, in particular autoimmune diseases, on the basis of the identification of marker sequences.
WO2010/000874, in the name of the applicant, for example describes the diagnosis of prostate carcinoma and prostate inflammation by means of a protein biochip and provides specific diagnostic marker sequences.
In this case, these marker sequences for the respective indications were able to be identified sensitively for the first time by means of protein biochips.
Protein biochips allow the advantageous assignment of individual high-density loci (spots) to the respective marker sequences and signals.
For example, a high-affinity binder from a sample can be added to a protein biochip and the interaction between a high-affinity binder and a marker sequence in an environment containing various marker sequences can be detected. This is likewise possible with an array, wherein marker sequences are arranged at individual spots (loci).
Diagnostic questions can also be dealt with in this way depending on the selection of marker sequences and the high-affinity binder used. In particular, diagnosis methods are of interest in which a test subject or patient provides a sample containing high-affinity binders and the sample is exposed to an array comprising selected marker sequences for the diagnosis of a question or of an event (for example condition, illness, etc.).
There is a high demand however to simplify such methods and to minimise, in particular miniaturise, such methods.
A plurality of marker sequences can generally be used for a specific diagnostic question, and what are known as “marker panels” are used for improved handling of diagnostic questions or events. For example, marker sequences known from the literature are combined with one another in the prior art such that increased validity can be achieved.